The blood-epididymal barrier creates a unique microenvironment critical for sperm maturation. There is little information on proteins comprising epididymal tight and adhering junctions or on factors regulating their expression. Claudins are a family of transmembrane proteins reported to be exclusively localized to tight junctions. In the present study the expression of claudin-l (Cl-1) was examined with respect to the different cell types of the epididymis and its various regions as well as its expression during postnatal development and regulation by testicular factors, using both immunocytochemistry and Northern blot analysis. RT-PCR of adult epididymal and testicular RNA (positive control) indicated that Cl-1 messenger RNA (mRNA) transcripts were present in all regions of the epididymis. In the adult, Cl-1 was localized immunocytochemically along the entire length of the lateral plasma membranes between adjacent principal cells, including apical areas containing tight junctions, as well as at the interface between principal and basal cells and along the basal plasma membrane of the epithelium in relation to the basement membrane. Northern blot analysis of adult epididymis with a rat Cl-1 complementary DNA indicated the presence of two hybridizing bands of 4.0 and 1.5 kb. Postnatally, in the caput-corpus and cauda epididymidis, mRNA levels for both transcripts were lowest on day 7. In the caput-corpus epididymidis, mRNA levels for the 1.5-kb transcript increased significantly between 7 and 14 days, whereas the levels of the 4.0-kb transcript were significantly higher by day 21. Postnatal studies revealed that in the initial segment and caput epididymidis, Cl-1 immunostaining was present along the entire length of the lateral plasma membranes of undifferentiated epididymal epithelial cells as early as day 7, including apical areas containing tight junctions. By day 21, staining was identical to that of adult animals, but as this is an age when androgen levels are not at their peak, the data would suggest that they are not a prominent factor regulating Cl-1 expression. Orchidectomy and orchidectomy plus testosterone replacement experiments revealed differences in Cl-1 immunostaining in the initial segment, suggesting that localization of Cl-1 in epididymal tight junctions is androgen dependant. Thus, Cl-1 expression in the initial segment appears to be only partially under the control of androgens. However, in all other epididymal regions, orchidectomy with or without testosterone replacement, revealed no changes to the normal staining pattern, suggesting that androgens do not regulate Cl-1 expression in these regions. Taken together, these studies demonstrate that Cl-1 expression in the epididymis is not localized exclusively to tight junctions, but appears along the entire interfaces of adjacent epithelial cells as well as along the basal plasma membrane, suggesting a role for Cl-1 as an adhesion molecule. The data also suggest that the regulation of Cl-1 in the epididymis is complex and multifactorial.
The effect of technology-enhanced learning (TEL) strategies in higher education has arguably been transformative despite the not-insignificant barriers existing in this context. Throughout the discourse very little attention has been paid to those primarily responsible for this implementation-academic teaching staff. This paper aims to highlight the impact of academic workload allocations, an often silent barrier to the uptake of TEL strategies in higher education. We will discuss the effects of academic identity and culture, preferential time allocation to associative activities, academic technological capacity, university policies and workload and funding models on the uptake, and implementation on TEL in higher education. Our aim is to highlight the risks to staff, students and institutions should these concerns not be addressed and to propose a model for utilisation by all staff responsible for implementing flexible workload models supportive of further implementation of TEL strategies across the sector.
In the epididymis, Cx43 forms gap junctions between principal and basal cells but not between adjacent principal cells. Cx30.3, 31.1, and 32 were identified in adult rat epididymis by RT-PCR, whereas Cx26 was present in young rats. Postnatal development studies indicate that Cx26 mRNA was detectable only in the caput-corpus region of the epididymis and that levels increased by fivefold during the first 4 wk postnatally, when epithelial cells differentiate, and decrease to nondetectable levels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout the epididymis in young rats and began to increase in the second and third weeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32 were localized to the lateral plasma membranes between adjacent epithelial cells of the epididymis. Colocalization studies indicate that Cx26 and Cx32 exist either independently of one another or can colocalize along the lateral plasma membrane of epithelial cells in young rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.
Although spermatozoa are formed during spermatogenesis in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical spermatozoal functions are acquired in the epididymis where a specific luminal environment is created by the blood-epididymal barrier; proteins secreted by epididymal principal cells bind to maturing spermatozoa and regulate the maturational process of the spermatozoa. In the epididymis, epithelial cell-cell interactions are mediated by adhering junctions, necessary for cell adhesion, and by tight junctions, which form the blood-epididymal barrier. The regulation of these cellular junctions is thought to represent a key determinant in the process of sperm maturation within the epididymis. Tight junctions between adjacent principal cells permit the formation of a specific microenvironment in the lumen of the epididymis that is essential for sperm maturation. Although we have made significant progress in understanding epididymal function and the blood-epididymal barrier, using animal models, there is limited information on the human epididymis. If we are to understand the normal and pathological conditions attributable to human epididymal function, we must clearly establish the physiological, cellular and molecular regulation of the human epididymis, develop tools to characterize these functions and develop clinical strategies that will use epididymal functions to improve treatment of infertility.
The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.
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